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Phospho-ERK (Thr202/Tyr204) Cellular Assay Kit

The trusted homogeneous cell-based assay for GPCR and RTK targeted screening as MAPK pathway readout

Fast track your research and speed up your time to consistent results - from basic research to high-throughput drug screening.  Cisbio’s homogeneous and robust HTRF® phospho-ERK assay kit is designed for the cell-based quantitative detection of ERK modulation, phosphorylated on Thr202/Tyr204. The simple mix-and-read protocol eliminates all wash steps for a faster analysis and easy miniaturization while maintaining a high quality and specific output.

 

Reproducible results - ideal for screening small molecule compounds or biologics directly in the cell, creating leads you can rely on. The phospho-ERK ready-to-use kit contains all the reagents you need and offers enhanced convenience over other immunoassay technologies. Let your research flow faster and more efficiently with Cisbio’s smart phospho-protein assays!

Features

  • High dynamic range for IC50 consistency and multi-parametric analysis of various readouts 
  • Excellent assay robustness, represented by high Z' scores, due to cryptate chemistry
  • Truly homogeneous, for faster analysis and easy miniaturization for HTS & profiling
  • High species cross reactivity: human, mouse, rat, dog, monkey, hamster & more
  • Low sample volume required and simple protocol

Applications

  • Cell-based functional assay- ideal for high throughput screening
  • MAPK pathway and RTK activity modulation
  • Monitors Gi, Gs, or Gq coupled receptor activation and β -Arrestin signaling readout
  • Mechanism of action of biotherapeutics
  • Oncology, cardiovascular, CNS, diabetes, inflammation, metabolic disorders, infectiology

HTRF - the homogeneous cell-based sandwich immunoassay

The phospho-ERK assay is based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-ERK1/2 antibodies, one labeled with Eu3+-cryptate (donor) and the other labeled with d2 (acceptor). Upon MAPK pathway activation, ERK1/2 is phosphorylated on Thr202/Tyr204. After phosphorylation, the phospho-ERK1/2 antibodies will recognize the phosphorylated residue, and the proximity of donor and acceptor will consequently lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed.

Cellulerk assay theory

The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step. The phospho-ERK assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho-ERK1/2 can be quantitatively detected using the HTRF phospho-ERK kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol:

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection of phosphorylated ERK1/2, lysates are subsequently transferred to a 384sv assay plate, where the HTRF reagents are added. This also enables the monitoring of cell viability and confluence in an appropriate cell culture plate.

Cellulerk two plate protocol

One-plate assay protocol:

This protocol can be further streamlined to a one-plate assay in which plating, treatment and detection are all performed in a single plate. No wash steps are required. Ideal for HTS, this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high quality and sensitive output.

Cellulerk one plate protocol

 

Cellulerk pathway

MAPK/ERK cell signaling

The MAPK/ERK signaling cascade is activated by a wide variety of receptors involved in growth and differentiation including growth factor receptors, like receptor tyrosine kinases RTKs, integrins, cytokine receptors, GPCRs  and ion channels. The core cascade Ras-Raf-MEK-ERK signal transduction leads to the regulation of a great variety of cellular processes including adhesion, cycle progression, migration, apoptosis/survival, differentiation, metabolism, proliferation, etc. ERK is a Mitogen-Activated Protein kinase that belongs to the family of serine/threonine kinases. MEK1/2 catalyze the phosphorylation of ERK1/2 at Tyr204 and then Thr202. Both phosphorylations are required for ERK1/2 activation. In response, ERK phosphorylates hundreds of cytoplasmic and nuclear substrates, including regulatory molecules and transcription factors. The wide complexity and diversity of MAPK signaling makes ERK a key regulator and major signaling node in biology. Signal transduction via MAPK/ERK is a highly conserved pathway, with extensive cross talk between other pathways and a wide variety of signal and stimuli responsiveness known today. More than 80 pathways in which ERK is involved are currently described (KEGG database). The activity of the Ras-Raf-MEK-ERK core cascade is increased in about one-third of all human cancers. Thus, inhibition of targets in the MAPK/ERK pathway represents an important anti-tumor strategy, whereas the assessment of ERK activation itself serves as a valuable ubiquitous pathway readout. Phospho-ERK is therefore an important tool for monitoring upstream dose-dependent modulation of endogenous or overexpressed targets.

ERK in GPCR signaling

ERK modulation plays a major role in GPCR signaling and is therefore an important measurable GPCR readout. GPCRs act via G proteins to regulate a wide range of cellular functions. Upon stimulation, these receptors activate effectors like adenylate cyclase and phospholipase C, which influence not only intracellular concentrations of second messengers like cyclic AMP, diacylglycerol, inositol 1,4,5 trisphosphate and Ca2+, but also mediate

ERK1/2 phosphorylation. Activation of Gai/o, Gas, Gaq/11 or Ga12/13 modulates ERK1/2 activation via numerous mechanisms. In addition, both alpha and beta subunits of G proteins can stimulate ERK1/2 phosphorylation through transactivation of receptor tyrosine kinases. GPCRs have also been shown to mediate ERK1/2 activation in a G protein-independent but beta-arrestin dependent manner.

For all therapeutics areas

  • Oncology
  • Diabetes/Metabolic
  • Neurology
  • Cardiovascular
  • Inflammation
  • Infectiology

 

1. Western Blot versus HTRF assay

A431 cells were grown in a T175 flask 37°C, 5% C02, 2days. Stimulation was done with 100 nM EGF for 10 min. After elimination of cell culture medium, 3 ml of supplemented lysis buffer was added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging.

Total protein concentration was determined by BCA assay: 20 106 cells lysed under 3 ml corresponds to 3.2 mg/ml of total proteins. (50 000 cells per well corresponds to 25 µg of total proteins) 16 µl of serial lysates dilutions were dispensed and analysed side-by-side by WB and HTRF assay.

The HTRF assay is 8-fold more sensitive than the Western Blot: 200 cells can be detected by using Phospho-ERK assay, while 1960 cells are needed for the Western Blot.

Cellulerk western blot graph

 

2. Gaq/i coupled receptor activation

Results obtained on CHO-CCR5 (25 000 cells) activated with Rantes & MIP-Iß for 10', using the two-plate assay protocol.

Gaq/i coupled receptor activation.

Cellulerk rgqi coupled graph

 

3. RTK activation: functional characterization of a cell line

Results obtained on cells expressing EGFR1, pre-treated or not with Cetuximab (therapeutic monoclonal antibody) or erlotinib (Tyrosine kinase inhibitor) for 2 hours at 37°C. Cells were then stimulated for 10 min with EGF.

The results show that the 2 compounds can efficiently inhibit ERK phosphorylation. They confirm that the monoclonal therapeutic antibody Cetuximab targets the EGFR1 binding site, and therefore prevents the activation of the ERK signaling pathway.

Cellulerk phospho erk inhibition graph

 

4. Screening robustness:

CHO-M1 (5,000 cells/well - 384-well small volume plate) were stimulated with Carbachol for 10 minutes at RT.

Z' values in fifty replicates were calculated between unstimulated cells (basal level) and 2 selected Carbachol concentrations, 1.3 µM and 4 µM, corresponding to the EC80 and EC100 respectively.

Assay was performed using the One-plate assay protocol on a robotic system from CyBio™ (CyBi®vario equipped with 384/25 µL pipeting head). Results were read on PHERAStar Plus (BMG Labtech).

Cellulerk screening robustness graph

Ordering Info

DescriptionCat. noProduct insertMSDS
ERK phospho-T202/Y204 kit - 500 tests64ERKPEG
ERK phospho-T202/Y204 kit - 10,000 tests64ERKPEH
ERK phospho-T202/Y204 kit - 50,000 tests64ERKPEI

Companion products

DescriptionCat. noProduct insertMSDS
ERK phospho-T202/Y204 kit control lysate62ERKTDA
-
Advanced ERK phospho-T202 /Y204 kit - 200 tests64AERPEF
-
Advanced ERK phospho-T202 /Y204 kit - 500 tests64AERPEG
-
Advanced ERK phospho-T202 /Y204 kit - 10,000 tests64AERPEH
-
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
-
ERK total kit - 500 tests64NRKPEG
-
ERK total kit - 10,000 tests64NRKPEH
-
ERK total kit - 50,000 tests64NRKPEI
-




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